Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35830
標題: 從竹嵌紋病毒複製蛋白質及其衛星核酸構成之複製蛋白質複合物中尋找宿主因子
To identify host factors involved in the replication complex of Bamboo mosaic virus replicase and its satellite RNA
作者: 林姿伶
Lin, Tzu-Ling
關鍵字: 本氏菸草
Nicotiana benthamiana
複製蛋白質複合物
竹嵌紋病毒
衛星核酸
銀染
免疫沉澱
replicase complex
Bamboo mosaic virus
satellite RNA
silver stain
immune-precipitation
出版社: 生物科技學研究所
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摘要: 竹嵌紋病毒 (Bamboo mosaic virus, BaMV) 為一正股 RNA 病毒,基因體全長約 6.4-kb,包含五個轉譯架構 (open reading frames, ORFs)。ORF1 可轉譯出病毒核酸複製酵素,從 N 端到 C 端分別為戴帽酵素活性區 (capping enzyme domain)、類解旋酵素活性區 (helicase-like domain) 及核醣核酸聚合酵素活性區 (RNA-dependent RNA polymerase, RdRp)。竹嵌紋病毒衛星核酸 (BaMV satellite RNA, satBaMV) 為 0.83-kb 之正股 RNA,目前已知 satBaMV 的複製需仰賴 BaMV。本實驗研究目的,是欲尋找與病毒複製有關的宿主蛋白,藉此了解病毒的複製機制。研究方法是以農桿菌 (Agrobacterium tumefaciens) 攜帶 satBaMV RNA 及 BaMV ORF1接種於煙草植物葉片,感染後一至五日分別收取葉片。植物萃取液經 30,000 × g 離心取沉澱之膜組織 (P30),以單獨感染 satBaMV RNA 葉片為對照組,結果顯示感染後第二天的複製酵素表現量最多。接著再以含 N-十二烷基肌氨酸鈉緩衝液從 P30 溶出含有複製酵素的蛋白質,藉由 RdRp 上的HA-tag 作為抗原進行免疫沉澱法 (immuno-precipitation)。經過梯度電泳 (Tricine SDS-4~13% PAGE) 與銀染呈色後,由液態層析串聯質譜儀 (LC/MS/MS) 分析,得到多個可能與 BaMV 相關的宿主因子,包含 DEAD-box ATP dependent RNA helicase 7-like, ATP-dependent DNA helicase Q-like 2-like, RNA helicase, exoribonuclease, vacuolar H+-ATPase B subunit, ripening-related protein, pleiotropic drug resistance like protein, putative S-adenosylmethionine synthetase, scarecrow-like protein 5, ClpA regulatory subunit of Clp protease complex, sucrose transporter, isocitrate dehydrogenase-like protein, MAP kinase phosphatase-like protein, disease resistance protein RGA2, calmodulin-binding protein-like protein。目前正以病毒靜默系統 (virus induce gene silence, VIGS) 的實驗觀察這些宿主因子對病毒與衛星核酸複製的影響。
Bamboo mosaic virus (BaMV) is a 6.4-kb positive-sense RNA virus, containing five open reading frames (ORFs). The ORF1 encodes a 155 kDa viral replicase, comprising a capping enzyme, a RNA 5’-triphosphatase and a RNA-dependent RNA polymerase (RdRp) from N- to C- terminus. BaMV satellite RNA (satBaMV) is dependent on BaMV for replication. The purpose of this study is to identify host factors involved in the replication of BaMV. Agrobacterium tumefaciens carrying BaMV replicase and BaMV-Satellite RNA-F4 expression plasmid was used for Nicotiana benthamiana plants infiltration. The leaves harvested at the different time points after infiltration were homogenized in extraction buffer, followed by centrifugation at 30,000 ×g to obtain the membrane fraction (P30). Western blot analysis of P30 showed that the virus replicase accumulated mostly at the second day. P30 was solubilized with sarkosyl. The solubilized proteins were collected by immune-precipitation using a HA-tag antibody and analyzed by SDS-PAGE. Compared to the control, the gel region having differential protein bands were excised and subjected to liquid chromatography-tandem mass spectrometry (LC/MS/MS). The results suggested several hypothetial host factors, including DEAD-box ATP dependent RNA helicase 7-like, ATP-dependent DNA helicase Q-like 2-like, RNA helicase, exoribonuclease, vacuolar H+-ATPase B subunit, ripening-related protein, pleiotropic drug resistance like protein, putative S-adenosylmethionine synthetase, scarecrow-like protein 5, AtClpC, sucrose transporter, isocitrate dehydrogenase-like protein, disease resistance protein RGA2,calmodulin-binding protein-like protein.The effects of these candidates on BaMV replication are being investigated by virus-induce gene silencing (VIGS).
URI: http://hdl.handle.net/11455/35830
其他識別: U0005-1208201316114300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1208201316114300
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